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Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open. [ABSTRACT FROM AUTHOR]

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE [ABSTRACT FROM AUTHOR]

Aspergilloglutamic peptidase from Aspergillus niger var. macrosporus (AGP) is one of the so-called pepstatin-insensitive acid endopeptidases, which are distinct from the well-studied aspartic peptidases. Among the known homologues of the glutamic peptidases, AGP is a unique two-chain enzyme with a light chain and a heavy chain bound non-covalently with each other, and thus is an interesting target for protein structure–function relationship studies. In this article, we report the crystal structure of a dimeric form of the enzyme at a resolution of 1.6 Å. This form has a unique structure in which the C-terminal region of the light chain of one of the molecules binds to the active site cleft of the other molecule like a part of a substrate. This form mimics the enzyme-activation product complex produced upon autoproteolysis, and provides a structural clue that could help to clarify the activation mechanism. This type of dimeric structure of a peptidase is here reported for the first time. [ABSTRACT FROM PUBLISHER]